DESCRIPTION: Macrophages (M) are crucial for human immunodeficiency virus type 1 (HIV) pathogenesis. In addition to their antigen presenting function, these cells appear to be important targets for HIV infection in extravascular tissues. Nearly all primary HIV strains are capable of replication in M, and the overwhelming predominance of macrophage-tropic (M-tropic) virus strains early in disease suggests that these cells may be involved in virus transmission, particularly for sexually acquired infection. Substantial work has been accomplished over the last decade to define molecular determinants of M-tropism. This includes identification of the HIV Env V3 loop as a critical domain for defining cellular tropism, and the identification of coreceptors, particularly the chemokine receptor CCR5, which is required for efficient entry of M-tropic HIV strains into M as well as CD4+T cells. Despite these impressive advances, specific mechanisms of entry, viruses that use CD4 and CCR5 (R5 strains) infect M more efficiently that those using CD4 and CXCR 4 (X4 strains). Efficiency of HIV infection may therefore involve cellular factors in addition to CD4 and chemokine receptors. During studies to identify additional cellular factors that could impact HIV entry, we found that a number of independently generated antibodies to a tetraspan cell surface protein, CD63, could block HIV entry into M, but not T cells. Although little is known about CD63, another tetraspan protein CD81 has been proposed as a receptor for HCV, and other tetraspan proteins have been implicated in signal transduction. Notably, four independently generated monoclonal antibodies specific for CD63 inhibited HIV infection of M, arguing against non-specific mechanisms of blockade. Thus CD63 could play a critical role in M-tropism. The proposed novel anti-retroviral therapies targeting HIV entry. Specific Aim 1. To further characterize anti-CD63 inhibition of HIV entry into M, we will assess strain dependence, and chemokine receptor usage (CCR5 or CXCR4) associated with this inhibition. We will confirm the impact of CD63 on multiple parameters, including receptor expression, conformation, and ability to support fusion/infection by introducing viral receptors into cellular backgrounds that are CD63 deficient, then introducing CD63 as well. Specific Aim 2. To identify mechanisms of CD63-mediated HIV entry, including a) CD63 binding to gp120, CD4 and CCR5, b) receptor/coreceptor interactions or co-localization, c) effects on CD4 turnover and d) signal transduction. Specific Aim 3. To characterize and define mechanisms of acquired HIV resistance to anti-CD63 inhibition. We will also assess potential synergy of anti-CD63 inhibition together with inhibition of chemokine receptor binding (AMD 3100 for CXCR4, as well as candidate CCR5 antagonists) and viral fusion (T-20), particularly for virus strains with resistance to these entry inhibitors.